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Abstract

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The
SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that
is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as
well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and
the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the
yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the
interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was
then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in
mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8–
ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our
findings suggest that ORF6 protein may play a role in virus replication

The successful determination of reliable protein interaction networks (PINs) in several species in the
post-genomic era has hitherto facilitated the quest to understanding systems and structural properties of
such networks. ...